Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases

Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 588.6 → 264.4 for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625–160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease.

Factors Associated with Changes in Bone Mineral Content among Girls in Early Pubertal Age

The study was conducted to investigate the factors associated with bone mineral content ( BMC) changes among early pubertal aged girls. Two hundred and thirty girls between 9 to 11 years of age participated in four surveys over 2 year period. During each survey, dietary intakes were collected with 3-day food records, BMC of left leg's calcaneus were measured by PIXI ( Lunar Ltd.) and body composition was measured by bioimpedance method ( Inbody 3.0 Biospace Co. Ltd, Seoul, Korea) . Mean values of BMC increased in consecutive measurements - 1.48 g, 1.54 g, 1.61 g and 1.66 g. The change of BMC between the first and last measurements was significantly positively correlated with % body fat, negatively correlated with BMC at the baseline ( p < 0.05) . Change of BMC was also significantly positively correlated with % change of lean mass and mineral mass between the first and last measurements. Nutrient intakes during the study period were calculated as mean daily intakes from all surveys. Mean daily intakes of Vit. C and Vit. B6 were positively and isoflavone intake was negatively correlated with changes of BMC ( p < 0.05) . In stepwise regression of BMC change with body composition and nutrient intakes, baseline values of BMC, weight, BMI and age, % change of mineral mass during the study period, mean intakes of isoflavone, Vit. B6, protein, carotene and zinc were significant explanatory variables ( R 2 = 0.38, p < .001, F = 32.39) . The results imply that the change of BMC among early pubertal aged girls are associated with some body composition and intakes of certain nutrients.